Disease Models & Mechanisms最新文献

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in MECP2, which encodes methyl-CpG-binding protein 2, a transcriptional regulator of many genes, including brain-derived neurotrophic factor (BDNF). BDNF levels are lower in multiple brain regions of Mecp2-deficient mice, and experimentally increasing BDNF levels improve atypical phenotypes in Mecp2 mutant mice. Due to the low blood-brain barrier permeability of BDNF itself, we tested the effects of LM22A-4, a brain-penetrant, small-molecule ligand of the BDNF receptor TrkB (encoded by Ntrk2), on dendritic spine density and form in hippocampal pyramidal neurons and on behavioral phenotypes in female Mecp2 heterozygous (HET) mice. A 4-week systemic treatment of Mecp2 HET mice with LM22A-4 restored spine volume in MeCP2-expressing neurons to wild-type (WT) levels, whereas spine volume in MeCP2-lacking neurons remained comparable to that in neurons from female WT mice. Female Mecp2 HET mice engaged in aggressive behaviors more than WT mice, the levels of which were reduced to WT levels by the 4-week LM22A-4 treatment. These data provide additional support to the potential usefulness of novel therapies not only for RTT but also to other BDNF-related disorders.

De novo truncating variants in fibrosin-like 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development, and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, also did not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.

Founder mutations are disease-causing variants that occur frequently in geographically or culturally isolated groups whose shared ancestor(s) carried the pathogenic variant. While some disease alleles may vanish from the genetic pool due to natural selection, variants with weaker effects may survive for a long time, thereby enhancing the prevalence of some rare diseases. These are predominantly autosomal recessive diseases but can also be autosomal dominant traits with late-onset or mild phenotypes. Cultural practices, such as endogamy and consanguinity, in these isolated groups lead to higher prevalence of such rare diseases compared to the rest of the population and worldwide. In this Perspective, we define population isolates and the underlying genetic mechanisms for accumulating founder mutations. We also discuss the current and potential scientific, clinical and public-health implications of studying founder mutations in population isolates around the world, with a particular focus on the Arab population.

P4-ATPases flip lipids from the exoplasmic to cytoplasmic leaflet of cell membranes, a property crucial for many biological processes. Mutations in P4-ATPases are associated with severe inherited and complex human disorders. We determined the expression, localization and ATPase activity of four variants of ATP8A2, the P4-ATPase associated with the neurodevelopmental disorder known as cerebellar ataxia, impaired intellectual development and disequilibrium syndrome 4 (CAMRQ4). Two variants, G447R and A772P, harboring mutations in catalytic domains, expressed at low levels and mislocalized in cells. In contrast, the E459Q variant in a flexible loop displayed wild-type expression levels, Golgi-endosome localization and ATPase activity. The R1147W variant expressed at 50% of wild-type levels but showed normal localization and activity. These results indicate that the G447R and A772P mutations cause CAMRQ4 through protein misfolding. The E459Q mutation is unlikely to be causative, whereas the R1147W may display a milder disease phenotype. Using various programs that predict protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments, suggesting that such analysis is useful in identifying protein misfolding disease-associated variants.

Interpreting the wealth of rare genetic variants discovered in population-scale sequencing efforts and deciphering their associations with human health and disease present a critical challenge due to the lack of sufficient clinical case reports. One promising avenue to overcome this problem is deep mutational scanning (DMS), a method of introducing and evaluating large-scale genetic variants in model cell lines. DMS allows unbiased investigation of variants, including those that are not found in clinical reports, thus improving rare disease diagnostics. Currently, the main obstacle limiting the full potential of DMS is the availability of functional assays that are specific to disease mechanisms. Thus, we explore high-throughput functional methodologies suitable to examine broad disease mechanisms. We specifically focus on methods that do not require robotics or automation but instead use well-designed molecular tools to transform biological mechanisms into easily detectable signals, such as cell survival rate, fluorescence or drug resistance. Here, we aim to bridge the gap between disease-relevant assays and their integration into the DMS framework.

Abnormal extracellular signal-regulated kinase 1/2 (ERK1/2, encoded by Mapk3 and Mapk1, respectively) signaling is linked to multiple neurodevelopmental diseases, especially the RASopathies, which typically exhibit ERK1/2 hyperactivation in neurons and non-neuronal cells. To better understand how excitatory neuron-autonomous ERK1/2 activity regulates forebrain development, we conditionally expressed a hyperactive MEK1 (MAP2K1) mutant, MEK1S217/221E, in cortical excitatory neurons of mice. MEK1S217/221E expression led to persistent hyperactivation of ERK1/2 in cortical axons, but not in soma/nuclei. We noted reduced axonal arborization in multiple target domains in mutant mice and reduced the levels of the activity-dependent protein ARC. These changes did not lead to deficits in voluntary locomotion or accelerating rotarod performance. However, skilled motor learning in a single-pellet retrieval task was significantly diminished in these MEK1S217/221E mutants. Restriction of MEK1S217/221E expression to layer V cortical neurons recapitulated axonal outgrowth deficits but did not affect motor learning. These results suggest that cortical excitatory neuron-autonomous hyperactivation of MEK1 is sufficient to drive deficits in axon outgrowth, which coincide with reduced ARC expression, and deficits in skilled motor learning. Our data indicate that neuron-autonomous decreases in long-range axonal outgrowth may be a key aspect of neuropathogenesis in RASopathies.